Process for producing l-glutamic acid by using bacteria



United States Patent 3,399,114 PROCESS FOR PRODUCING L-GLUTAMIC ACID BY USING BACTERIA Takeyoshi Olisawa, Mitsuru Shibukawa, and Hideomi Takahashi, Nobeoka-shi, Japan, assignors to Asahl Kasei Kogyo Kabushikikaisha, Osaka, Japan No Drawing. Filed Aug. 16, 1965, Ser. No. 480,09 Claims priority, application Japan, Aug. 22, 1964, 39/ 47,663 11 Claims. (Cl. 19547) ABSTRACT OF THE DISCLOSURE A process for accumulating L-glutamic acid by inoculating a biotin-requiring L-glutamic acid-producing bacterium selected from the group consisting of Microbacteriumflavum var. glutamicum, Brevibacterium genus, Brevibacterium dibalicutum, Micrococcus genus, Micrococcus glutamicus, and Corynebacterium genus into a culture medium containing molasses containing an excess of biotin as a main carbon source, by adding a polyoxyethylene fatty acid ester type surfactant into said medium at the initial stage of the logarithmic growth phase of said bacterium and by further adding an alkylamine salt type cationic surfactant into the medium at the proper time of between the middle and the end stages of the logarithmic growth phase of said bacterium.

The present invention relates to a process for producing L-glut-amic acid comprising culturing bacteria in a carbohydrate culture medium containing an excess of biotin as the main carbon source and more particularly the invention concerns an advanced process for producing L-glutamic acid.

The object of the present invention is to provide an industrially practicable method for producing L-glutamic acid by a fermentation process characterized by innoculating a fermentation medium containing a substance having an excess of biotin, for example, cane sugar waste molasses, or beet sugar waste molasses with an excess of biotin added, as the main carbon source, with biotinrequiring, L-glutamic acid-producing bacterium, culturagents have very little or an unpredictable effect on the yield of L-glutamic acid in an industrial scale of production,vond therefore these processes are almost of .no use in the industrial production of L-glutamic acid. Furthermore, even when a non-ionic surface active agent is used, the yield of said acid is greatly influenced by the amount of biotin in the medium. The yield of L-glutamic acid is decreased in proportion to the amount of biotin present in the medium no matter how much surface active agent is added. Of all the well known non-ionic surface active agents, only several have been found to be useful in the production of L-glutamic acid. Further, it has been assumed that, when these effective non-ionic surface active agents are used alone, the biotin content in the culture medium must be below a definite level such as less than about 20-30 g/l. for the industrial production of L- glutamic acid. However, if cane sugar waste molasses is added to the culture medium, for example, in an amount of 7% by weight (as total sugar), the biotin content in the culture medium will be more than 100 ug./l., and therefore such a material has never been utilized as a carbon source in the industrial scale production of said acid up to the present time.

The present inventors, after extensive research, have found that, contrary to expectations based on conventional thought, the addition of specific combinations of non-ionic surface active agent and cationic surface active agent to the medium is very effective in accumulating a large amount of L-glutamic acid in a medium containing biotin in excess.

The most effective surface active agents for use in the method of this invention are non-ion type agents such as polyoxyethylene monostearate, polyoxyethylene monopalmitate, polyoxyethylene monomy-ristate and the like, and cationic type surface active agents such as de'cylamine, laurylamine, myristylamine, palmitylamine, and the like. As described hereinabove, these surface active agents are almost useless when they are employed alone in the production of L-glutamic acid on an industrial scale, since the yield of L-glutamic acid is very low or uncertain. These facts are made clear from the following test results shown in Tables 1 and 2.

TEST 1 TABLE 1.EXPERIMENTAL CONDITIONS (COMPOSITIONS OF MEDIUM) Fermentation medium Seed medium,

g./dl. For A-method, For B-method,

g./dl. g./dl.

Cane sugar waste molasses (as a total sugar)- 4. 0 7. 0 7. 0 KHzP O4 0. 2 0. 2 0. 25 MgS04-7H2O---- 0.05 0.05 0. 05 FeS04-7Hz0- 0. 005 0.005 0. 0005 MllSOa-nHzO-. 0 0 0.0005 (NI-102304 0 0 0. 05 Urea 0. 8 1. 5 0 pH, after sterilization 7. 2 7. 2 7. 0

ing said bacterium aerobically while submerged and under constant agitation, adding a compound selected from polyoxyethylene fatty acid ester type nonionic surface active agents to the medium at the initial stage of the logarithmic growth phase of said bacterium, and further adding the second additive consisting mainly of alkylamine salt type cationic surface active agent thereto at the proper time of between the middle and the end stages of the logarithmic growth phase of said bacterium to increase the accumulation of L-glutamic acid in the medium.

Various studies have reported on L-glutamic acid fermentation employing non-ionic surface active agents, and also on fermentation processes employing cationic or anionic surface active agents. However, in general, except for the non-ionic surface agents, such surface active A-method.-Corynebacterium melassecola AS B-1285 (ATCC 17965) was cultured in a seed medium having the composition shown in Table 1 at 30 C. for 14 hours with agitation, and 1.5 ml. of the culture thus obtained was inoculated into ml. of said fermentation medium charged in a 500 ml. flask. Culturing was then effected at 30 C. for 48 hours with shaking. This method is hereinafter referred to as Flask Culture Test" or A- method.

B-method.C0rynebacterium melassecola AS B-1285 (ATCC 17965) was cultured in the seed medium shown in Table 1 in the same manner as described in A-method, and 400 ml. of the culture thus obtained was inoculated into 10 l. of the fermentation medium charged into a 201. jar fermentor. Culturing was then effected at 33-35 C. for 40 hours under aerobic conditions with agitation while keeping the pH of the medium at 7.0 by introducing 'ammonia gas. When the sugar concentration in the jar decreased to 1.5 g./dl., a sterilized solution of cane sugar waste molasses, the total sugar content of the solution being adjusted to 45 g./dl., was added to the medium to keep the concentration of sugar in the medium at around 1.5 g./dl. This method is hereinafter referred to Jar Fermentor Test or B-method.

TABLE 2.-AMOUNTS (g./dl.) OF L-GLUTAMIC ACID ACCUMULATED Amount A-method B-method Additive added Time of addition (Flask) (Jar) (A) lolyoxyethylene 1110110- 0.10 O.D.==0.35

palmitate ester. 0.30 O.D.=0.35 0. 4 hrs. after cultiv 0. do

(B) Myristylamine acetate... 0.035 O.D.=O.65

0. 042 O.D.=0.65 0. 010 10 hrs. after cultivation. 0.020 do (AH-(B) 0.10 O.D.:0

Tween (polyoxyethylene sorbitan monopalmitate).

In Table l, the time for addition was determined by checking the optical density (abbreviated as CD.) of the culture medium by means of a photocell colorimeter to follow-up the growth stage of fermentation bacteria. More particularly, a small sample of culture medium was taken out periodically, diluted with water to 20 times in volume, and optical density of the diluted medium was determined by means of a photocell colorimeter at 660 m in 10 mm. cell. Said value varies somewhat with the colorimeter employed, but, this variation may be compensated for checking each growing period beforehand with the same meter in common use, deducting the figure for the culture-initiating time from each of the measured values, and then using the thus obtained net values as standards for determining the optimum addition time for each additive.

The above table indicates that there are no particular differences in results obtained by the method of this invention and by the conventional method in the Flask Culture Test. However, in the Jar Fermentor Test (or with any other container bigger than the jar fermentor) in which aerating and agitating conditions different from those used in the flask test are employed an important difference is clearly observed. That is, these results point to an outstanding characteristic on an industrial scale of practice. This point is very important, since the scaling up in actual production of L-glutamic acid has been believed to be the most troublesome question in the fermentation industry.

The inventors have also studied the use of beet sugar waste molasses as the main raw material in the production of L-glumatic acid. The employment of beet sugar waste molasses as a main carbohydrate source can contribute an increase in the yield of product and also a decrease in the production cost, so if said material can be used for the production of L-glutamic acid it is advantageous. However, it should be pointed out that when this material is used as a carbohydrate source the fermentation conditions are considerably different from those of the conventional process employing glucose as a carbohydrate source.

The main reason why beet sugar waste molasses requires different fermentation conditions from glucose is that, if used under the same conditio s 2 5 gluCOse, the

ionic surface active agent to the medium. This finding is described in detail in the specifications of Japanese patent application Nos. 37/3l,4S8/l962 and 37/46,954/l962 (corresponding to French Patent No. 1,371,926). The exact role of the non-ionic surface active agent in the accumulation of L-glutamic acid is not yet clear, but it must have some connection with the amounts of nutrient factors in the medium. In a nutrient medium containing an excess of biotin, even if a large amount of non-ionic surface active agent is used, the yield of L-glutamic acid is still too low to be of practical use.

However, the inventors, after further study of said process, found that said fermentation can be carried out in a short period of time with a constant and a high yield of product by employing a combination of non-ionic surface active agents. That is, L-glutamic acid producing bacteria are cultured in a beet or cane sugar waste molassescontaining medium intentionally including an excess of biotin, and a polyoxyethylene fatty acid ester selected from the non-ionic surface active agents, and, subsequently, an alkylamine or salt thereof selected from the cationic surface active agents are added to the medium. By this method, the inventors have succeeded in obtaining a yield far superior to that obtained in the conventional process using a glucose or a beet sugar waste molasses medium. The remarkable effect obtained by this invention can fully be understood from the data shown in Table 3, which shows the correlation between the amounts of biotin added to the medium and the amounts of L-glutamic acid produced therein.

TABLE 3.-EFFECT OF BIOTINADDITION L-glutamic acid produced (g., dl.)

Tested bacterium Biotm added (,ug. ll.)

0. melassccola M. amnogiaph ilum,

TEST 2 (1) Media composition Seed Fermentation medium medium Beet sugar waste molasses (as total 4g./dl 7g./dl.

sugar). KHzPOi 0.25 g./dl.- 0.2 g./dl. MgSO4-7HZO 0.05 g./dl 0.05 g./dl. Urea 0.8 g./dl Biotin 5 g./dl.. FOSOi-HHzO and MnSOr-nHz Each 0.0005 g./dl. (NHi)zSO4 0.05 g./dl. pH adjusted after sterilization to 7.2 7.2. i

(2) Seed culturing Corynebacterium m'elassecola AS B-1285 (ATCC 17965) was inoculated into the seed medium and cultured at 30 C. for 14 hours with shaking.

(3) Fermentation C-method.75 ml. of fermentation medium having the above composition except that 0.05 g./dl. of (NH SO was replaced by 1.5 g./dl. of urea and containing biotin in an amount as shown in Table 4 below, was charged into a 500 ml. Sakaguchi flask and inoculated with 1.5 ml. of the seed culture obtained (2) above. Fermentation was then effected at 30 C. for 48 hours with shaking.

D-method.l0 l. of the above fermentation medium containing biotin in an amount as shown in Table 4 was charged into a 20 1. jar fermentor andinoculated with 400 ml. of the seed culture obtained from (2) above. Fermentation was then effected at 33-35 C. for 40 hours under aerobic conditions with agitation while keeping the pH of the medium at 7 by introducing ammonia gas. During fermentation, when the amount of sugar initially charged decreased to 1.5 g./dl., a sterilized beet sugar waste molasses solution, the total sugar being adjusted to 45 g./dl., was added to the medium thereby. keeping the concentration of sugar thereof at about 1.5 g./dl.

The results are shown in Table 4.

From the results shown in Table 4, it is apparent that there is no significant difference in the amount of L-glutamic acid between the method of this invention and the conventional method in fiask scale fermentation, but an outstanding difference is clearly observed in jar fermentor scale fermentation. Furthermore, with the method of this invention, it is possible to get a higher yield in a shorter period than with the conventional methods. Another advantage of this invention is that the yield is uniform and steady.

Briefly, in the fermentation of L-glutamic acid in a waste molasses-containing medium, this invention is characterized in that the culturing is started with an excess of biotin in the medium, and thereafter a component selected from polyoxyethylene fatty acid ester type nonionic surface active agents is added to the medium, and, after additional culturing, a component selected from alkylamine salt type cationic surface active agents is also added to the medium.

The surface active agents to be employed in the method of this invention are selected from the polyoxyethylene fatty acid esters and alkylamines commercially available. The preferred polyoxyethylene fatty acid esters are made up of fatty acids of C -C and polyoxyethylene parts having average molecular weights of 200-1500; the preferred alkylamines contain alkyl groups of 10 to 18 carbon atoms.

The correlation between amounts of said surface active agents to be added and the time of addition is such that a proportionately higher amount is required in proportion to the lapse of time. A medium with a pH of 6-9 is satisfactory, optimum results being obtained at 7-8. Polyoxyethylene fatty acid esters and alkylamines may preferably be added to the medium in an amount of 0.05-0.2 g./dl. and 0.0050.05 g./dl., respectively, on the basis of the medium volume prior to the inoculation of the bacterium. In accordance with the present invention, the first additive (polyoxyethylene fatty acid esters) is added to the medium at any time between the inoculation of bacterium and the middle stage of logarithmic growth phase of the bacterium, and the second additive (alkylamines) is added at any time between the middle stage and the later stage of said logarithmic growth phase of the bacterium employed. Though the time duration between the first and the second additives may somewhat vary with the type of bacterium, sugar waste molasses, and the conditions for aerating and shaking employed in the fermentation, said duration is, in general, within from 30 minutes to 8 hours. The growth of bacterium during the fermentation may easily be determined by measuring O.D. value of the medium TABLE 4 L-glutamic acid Amount Biotin accumulated (g./d1.) Additive added Time of addition (hour). (pg/1.) Note C-method D-method (Flask) (Jar) 0. 1o rum 4 hrs. from the beginning..- (A)+(B).; 0 3.07

0.010 After 10 hrs (A)+(B) 1 100 3. 54 Method of present invention.

. 0.10 0.D.=0.30 0 3 10 A B 0.030 O.D.=0.65 (AH-(B) 100 9. 61 Method of present invention.

0. 10 Initia1 v 3. 47 (C) 0. 10 After 4 hrs 3.

itial 6. 87 (C) 0. 10 0 Conventional method. O.D.=0 35 6.98

2.89 C Initial i 0.50 3. 21

0. l6 1. (C) }Initia1 NOTE.-O.D. has the same meaning as defined in Table 2 of Test 1. (A)=Polyoxyethylene monopalmitate; (B) =Laurylarnine acetate; (C) =Tween 40 (Atlas Powder 00.): polyoxyethylene sorbltan monopalmitate.

Ihid.

by means of a photocell colorimeter as hereinabove described.

The L-glutamic acid producing bacteria employed in the method of this invention are any of those hitherto known for the production of said acid, which may include:

Microbacterium genus-Microbacterium flavum var.

glutamicum Brevibacterium genus-Brevibacterium dibalicutum Micrococcus genusMicrococcus glutamicus Corynebacterium genus Corynebacterium lilium Corynebacterium carnae Corynebacterium melassecola and the like. Other than those listed above, any L-glutamic acid-producing bacteria which require biotin as a growth factor may satisfactorily be employed in this invention. The carbohydrate sources utilized in the present process are waste molasses such as cane and beet sugar waste molasses. They are used alone, or, if desired, in an admixture with other carbohydrate sources such as glucose, sucrose, starch hydrolyzate and hydro]. If the waste molasses is used with other sources given above, the amount of the waste molasses in the admixture must be at least by weight of the total of the total sugar. When the beet sugar waste molasses is used, biotin must be added to the medium in an amount of more than 'y/ 1.

The preferred nitrogen source used in the fermentation of this invention is urea and ammonia.

Inorganic salts required by bacterium, for example, phosphate, magnesium salt and manganese salt, may be added to the medium. The amount of such salts will depend on the type of molasses employed. Said carbohydrate source may be fed into the medium at one time or fractionally thereby maintaining the optimum concentration thereof during the course of fermentation. However, when Corynebacterium melassecola ATCC 17965 and 17966 is used, fractional feeding of the carbohydrate source is preferred.

As described hereinbefore, the present invention is particularly useful in the industrial scale of production of L-glutamic acid employing a submerged culture method under aerobic and agitation conditions, and is useful for eliminating the fluctuation in the yield of L-glutamic acid caused by the change in the quality of the molasses. Thus, the invention may contribute much to the L-glutamic acid fermentation industry from the standpoints of a constant yield, increased labor efiiciency and the like.

Fermentation is preferably carried out at a temperature of 25 to C. and is usually complete within 30-60 hours. When the fermentation is conducted aerobically while submerged, and under constant agitation, the ratio of L-glutamic acid to starting sugar reaches almost 60% within 30-38 hours and, at this point, the concentration of L-glutamic acid in the medium reaches as high as 8- 9.5 g./dl. After completing of fermentation, the L-glutamic acid may be separated from the medium by using a conventional method such as, for example, a sequence of steps of removing bacterial cells from the fermentation broth, concentrating the broth, adjusting the pH of the broth to 3.2 with hydhochloric acid, and leaving the concentrated broth in a refrigerator to precipitate the crude crystals of L-glutamic acid. The thus obtained crude crystals may be recrystallized to get pure L-glutamic acid crystals.

The invention is illustrated by the following examples, wherein Example 1 is an example using the prior art Microbacterium ammoniaphz'lum.

.. Example l.-Four hundred milliliters of seed culture obtained by culturing Micrabacteriumammoniaphilum ATCC 15354 aerobically in the following seed medium at 30 C. for 8 hours under submerged conditions were inoculated into 10! of the following fermentation medium (about 98 ,ug./l. biotin content) placed in a 20 1. jar fermentor.

COMPOSITIONS OF MEDIUM Made in Java Island, biotin and total sugar contents were 143 ug/ g. and 52 g./100 g., respectively. After inoculation of said seed into fermentation medium, the bacterium was cultured aerobically at 30 C. for 36 hours while submerged and under agitation, the pH of the medium being kept at 7 by introducing ammonia gas therein. About 4.5 hours later from the beginning of the cultivation (initial stage of logarithmic growth phase, i.e. at the time when the net O.D. value of the fermentation medium reached 0.35), the first additive previously sterilized Nonion P-6 (trade name: Nippon Oil and Fat Co. Ltd.) consisting mainly of polyoxyethylene monopalmitate was added to the fermentation medium in the concentration of 0.10 g./dl., and about one hour later, i.e. at the time the net O.D. value reached 0.65, the second additive previously sterilized Acetamin 24 (trade name used by Kao Soap Co., Japan) consisting mainly of lauryl amine acetate was added to the medium in the concentration of 0.030 g./d1. A mixed sugar-supplying solution consisting of 25 g./dl. cane sugar waste molasses (as total sugar) solution and 25 g./dl. glucose solution was added, as necessary, so as to maintain the sugar concentration in the medium at about 1.5 g./dl. After completing fermentation, the amount of L-glutamic acid in the fermentation broth was 8.89 g./dl. The fermentation broth thus obtained was then filtered to remove bacterial cells therefrom, concentrated from 12 1. to about 3 l., and adjusted to pH 3.2 by adding hydrochloric acid. By using the so called isoelectric point method, 905 g. of crude L-glutamic acid crystals were obtained.

Comparative tests employing only either the first additive or the second additive were also conducted in the same manner, and in these tests, the rate of L-glutamic acid accumulation was found to be 0.72 g./dl. or 2.78 g./dl. respectively.

Example 2.-Corynebacterium lilium NRRL B-2243 was cultured in a seed culture medium in accordance with the procedure described in Example 1, and the seed thus obtained was inoculated in a fermentation medium having the following composition.

The same molasses as described in the foregoing Exam-- ple 1 was used.

The bacterium was then cultivated aerobically at 30 C.

' which submerged and under constant agitation as described in Example 1.

After 24 hours of cultivation, the amount of L-glutamic acid in the medium was in the level of 5.06 g./dl. In this example, when the OD. reached 0.30, i.e. the initial stage of logarithmic growth phase of said bacteria, the first additive, a surface active agent consisting mainly of polyoxyethylene monomyristate, was added to the medium in a concentration of 0.1 g./dl., and reached 0.60, i.e. the mid dle stage of the logarithmic growth phase, the second additive, a surface active agent consisting mainly of C alkyd amine hydrochloride, was added to the medium at the rate of 0.023 g./dl.

Corynebacterium melass cola AS B-1285 (ATCC 17965) was cultured as described in Example 1, and 400 ml. of the resulting seed culture was added to l. of the fermentation medium having the following composition placed in a 1. jar fermentor,

Composition of fermentation medium: G./dl.

Cane sugar waste molasses (as total sugar) 7.0 KH PO 0.25 MgSO.,,- 7H O 0.05 FeSO -4H O 0.0005 M11804 (NH.,) 80 0.05

The bacterium was then cultivated aerobically at 3335 C. for hours while submerged and under agitation, while the pH of the medium being kept at 7.0 by introducing ammonia gas therein. When the OD. value of the culture medium reached 0.35, the first additive, separately sterilized Nonion P-6 (trade name used by Nippon Oil and Fat Co.) consisting mainly of polyoxyethylene monopalmitate, was added to the medium in the concentration of 0.1 g./dl., and when said O.D. value reached 0.65 the second additive, another surface active agent consisting mainly of myristyl amine acetate, was added to the medium at the rate of 0.038 g./dl. During the cultivation of said bacterium, a sugar-supplying solution containing cane sugar waste molasses g./dl. total sugar content) was added to the medium so as to maintain the sugar content at about 2 g./dl. After fermentation, the amount of L-glutamic acid in the fermentation broth was 9.41 g./dl.

Example 4.The same procedure as described in Example 1 was repeated but employing Corynebacterium melassecola AS B-l285 (ATCC 17965) in place of Microbacterium ammoniaphilum. In this case, 0.1 g./d-l. of the first additive, Nonion P6 consisting mainly polyoxyethylene monopalmitate, was added to the medium when the OD. value reached 0.25, and 0.03 g./dl. of the second additive, a surface active agent consisting mainly myristylamine butyrate, was added when the OD. value reached 0.70. After 36 hours fermentation, the amount of L-glutamic acid in the fermentation broth was 6.25 g./dl.

Example 5.Brevibdcrerium dibalicutum NRRL B- 2311 was cultured aerobically in the seed medium having the following composition at 30 C. for 8 hours while submerged and under agitation. Four hundred milliliters of the resulting seed culture were then inoculated into 10 l. of the fermentation medium having the following composition (54.4 g. biotin content) placed in a 20 1. jar fermentor, and the bacterium was cultured areob-ically at 30 C. for 36 hours while submerged and under agitation, the pH of the medium being kept at 7.0 by introducing ammonia gas.

COMPOSITION OF THE MEDIA Biotin and total sugar contents of the molasses were 6.6 g./100 g.

and 52 g./100 g., respectively.

About 4.5 hours from the beginning of said cultivation (initial stage of logarithmic growth phase of bacteria), i.e. at the time when the net O.D. value of said fermentation medium reached 0.30, the first additive, previously sterilized Nonion P-6 (trade name used by Nippon Oil and Fat Co., Japan) consisting mainly of polyoxyethylene monopalmitate, was added to the medium at the rate of 0.1 g./d'l., and about 1 hours later, i.e. at the time when the OD. value reached 0.65, previously sterilized Acetamine 24 (trade name used by Kao Soap Co., Japan) consisting mainly of laurylamine acetate was added in an amount of 0.030 g./dl. thereto. When the sugar concentration of culture medium decreased to 1.5 g./dl., a mixed sugar-supplying solution containing 25 g./dl. beet sugar waste molasses (as tot-a1 sugar) and 25 g./dl. glucose was continuously supplied to the fermentation medium to keep said sugar level in the medium. After fermentation, the amount of L-glutamic acid in the fermentation broth was 8.43 g./dl. The resulting broth was then filtered to remove off the bacterial cells, concentrated from 12 l. to about 3 l. and adjusted the pH of the concentrated filtrate to 3.2 by adding hydrochloric acid. By using the so-called isoelectric point method, 901 g. of crude L-glutamic acid crystals was obtained.

Comparative tests employing only either the first or second additive of said surface active agent showed poor results: the test in which only the first additive was added resulted in the yield of 2.73 g./dl. of L-glutamic acid and the test in which only the second additive was added resulted in the yield of 2.54 g./ d1. of L-glutamic acid.

Example 6.--L-glutamic acid fermentation was carried out in accordance with the procedure described in Example 5 but employing Corynebwcterium melassecola AS B-4821 (ATCC 17966) in place of Micr bacterium ammoniaphilum. In this case, polyoxyethylene monostearate as the first additive was added to the medium in an amount of 0.1 g./dl. when the OD. value of the medium reached 0.30, and C alkylam-ine hydrochloride as the second additive was added to the medium in an amount of 0.035 g./dl. when the OD. value of the medium reached 0.65. After 36 hours fermentation, the amount of L-glutamic acid in the fermentation broth was 8.73 g./dl.

Example 7.--L-glutamic acid fermentation was carried out in accordance with the procedure described in Example 5 but employing Brevibacterium lactofermentum ATCC 13869 in place of Brevibacterium dibalicutum. In this case, a fermentation medium having the following composition was used.

Composition of fermentation medium:

Beet sugar waste molasses (as a total sugar) g./dl 7.0 Biotin ,ug./dl 30 KH PO g./d1 0.2 MgSO -7H O g./dl 0.05 FeSO -4H O g./dl 0.0005 MnSO -4H O g./dl 0.0005 (NH SO g./dl 0.05

In this case, the surface active agent, polyoxyethylene monomyristate was added to the medium in an amount of 0.1 g./dl. when the OD. value reached 0.25 and C alkylamine hydrochloride was added to the medium in an amount of 0.032 g./dl. when the OD. value reached 0.65, respectively. Sugar-supplying solution employed in this example contained beet sugar waste molasses in an amount of 45 g./dl. as a total sugar. After 40 hours fermentation, the amount of L-glutamic acid in the fermentation broth was 7.62 g./dl.

Example 8.L-glutamic acid fermentation was carried out in accordance with the procedure described in Example 1 but employing Micrococcus glutamicus ATCC 13058 in place of Microbacterium ammoniaphilum, and a culturing temperature of 33 to 35 C. In this case, 0.10 g./dl. of polyoxyethylene monomyristate was added to the medium when the OD. value of the medium reached 0.30 and 0.025 g./dl. of myristyl amine acetate to the medium when the OD. value reached 0.60. After fermentation,

the second additive I 1 1 the amount of L-glutamic acid in the fermentation broth was 8.01 g./dl.

What is claimed is:

1. A process for producing L-glutamic acid by using bacteria which comprises inoculating biotin-requiring L- glutamic acid-producing bacterium selected from the group consisting of Microbacterium flavum var. glutamicum, Brevibactcrium genus, Micrococcus genus, and Cornynebacterium genus into a culture medium containing, as a main carbon source, a compound selected from the group consisting of (1) beet sugar waste molasses plus synthetic biotin, the biotin being excess of the amount required for the maximum growth of said bacterium, and (2) cane sugar waste molasses, culturing said bacterium aerobically while submerged and agitation conditions, adding a polyoxyethylene fatty acid ester type surface active agent to the medium at the initial stage of logarithmic growth phase of said bacterium and an alkylamine salt type surface active agent between the middle and the end stages of the logarithmic growth phase of said bacterium, and recovering the thus produced L-glutamic acid from the medium.

2. A process according to claim 1, wherein said culturing is conducted at a pH of 6 to 9.

3. A process according to claim 1, wherein said polyoxyethylene fatty acid ester type surface active agent is at least one compound selected from the group consisting of polyexyethylene monostearate, polyoxyethylene monopalmitate and poloxyethylene monomyristate.

4. A process according to claim 1, wherein said alkylamine type surface active agent is at least one compound 12 a selected from the group consisting of decylamine, laurylamine, myristylamine and palmitylamine.

5. A process according to claim 1, wherein said polyoxyethylene fatty acid ester type surface active agent is added to the medium in an amount of 0.05 to 0.2 g./dl.

6. A process according to claim 1, wherein said alkylamine type surface active agent is added to the medium in an amount of 0.005 to 0.05 g./ d1.

7. A process according to claim 1, wherein said medium contains more than /1. of biotin.

8. A process according to claim 1, wherein said culturing is conducted at a temperature between 25 and C.

9. A process according to claim 1, wherein the biotinrequiring L-glutamic acid-producing bacterium used for inoculating is Microbacterium flavum var. glutamicum.

10. A process according to claim 1, wherein the biotinrequiring L-glutamic acid-producing bacterium used for inoculating is Brevibacterz'um dibalz'cutum.

11. A process according to claim 1, wherein the biotinrequiring 'L-glutamic acid-producing bacterium used for inoculating is Micrococcus glutamicus.

References Cited UNITED STATES PATENTS 3,136,702 6/1964 Oltumura et al. -47 3,138,540 6/1964 Okada et a1 195-47 3,212,994 10/1965 Kono et al. 19529 3,338,793 8/1967 Yamamoto et al 195-47 LIONEL M. SHAPIRO, Primary Examiner. 

